The present invention provides a novel process for preparation of a mixture of 19 hydroxyelcosatetraenoic acid (19, HETE) and 20 hydroxyeicosatetracnoic acid (20 HETE).
Arachidonic acid metabolites obtained from three enzymatic pathways are known to be vasocative. A variety of cytochrome P450 metabolites affect vascular tone, including the xcfx89-hydroxylate products 19 HETE and 20 HETE. 20 HETE derived from arachidonic acid is released from activated neutrophilis and contribute to vascular tone, in number of organ systems. Zou et al. in 1994 have reported that inhibitors of renal vascular 20 HETE production impairs autoregulation of blood flow. Ma in 1993 have shown that 20 HETE is an endogenous vasoconstrictor of canine renal arcuate arteries, where as Escalante in 1993 has shown 20 HETE as an endothelium dependent vasoconstrictor in rabbit arteries. Pratt et al. in 1998 have reported 20 HETE is a potent vasodilator of bovine coronary arteries. It also contributes to vascular tone in a number of organ systems, such as aorta, mesentric, cortical and renal arteries. They also report that bovine arteries when incubated with 20 HETE produce prostacyclin in response to increasing concentration of 20 HETE. Furthermore, 20 HETE was shown to activate MAPK (mitogen activated protein kinase) which amplifies CPLxcex92 (cytosolic phosopholipase xcex92) activity and releases additional arachidonic acid by positive feed back mechanism which might play a role in signaling processes involved in inflammation, in cell growth, proliferation and differentiation. Schwartzmann in 1988 has shown 19(s) HETE may contribute to the regulation of renal function by regulating Na+xe2x80x94K+ ATPase which is essential for transtubular transport processes. There are no reports till date for microbial transformation of 20 HETE and 19 HETE from extraneously added arachidonic acid. The reports of 20 HETE production are either by incubating arachidonic acid with mammalian cells or by totally synthetic forms. Most of the reported transformation involve oxidation of activated carbon (allylic), but oxidation of unactivated carbon is very difficult even by chemical methods. This is due to lack of reactivity at this terminal carbon. One of the chemical methods for production of 19 HETE described by Schwartzmann et al., 1988 is as follows to a vigorously stirring xe2x88x9240xc2x0 C. solution of methyl 14-15 DHET (130 mg 0.369 mmole) in dichloroethane (4 ml) were added powdered, anhydrous sodium bicarbonate (40 mg, 0.387 mmole) and lead tetracaetate (171.6 mg 0.387 mmole). After 20 minutes the reaction mixture was passed rapidly through a silica gel bed and the filter cake was washed with dry ether (10 ml). The combined organic filtrates were concentrated under reduced pressure on a rotary evaporator. The resultant oily aldehyde was used directly in the next reaction after drying azeolropically with benezene n-Butyllithium (0.42 ml) was added dropwise with stirring to a xe2x88x9278xc2x0 C. solution 5 (R)-(t-butyldiphenyl sililoxy) hexyltriphenylphosphonium bromide in anhydrous THF (4 ml) under argon. After 45 minutes, a THF (2 ml) solution of above aldehyde was added slowly followed after 2 minutes by dry hexamethylphosporamide (1.5 ml). The reaction mixture was warmed over 0xc2x0 C., poured into 25% aqueous ammonium acetate and extracted with ethyl acetate (0.4xc3x9710 ml). The combined organic extracts were washed with water, brine and dried over sodium sulphate. Concentration and purification of the residue was done on silica gel colurnn. Yield was approximately 37%.
Biological methods require mammalian cells which are tedious and expensive, and the productivity is very low. One of the methods described by Escalante in 1989 is as follows: 20 HETE is prepared by incubating rate renal cortical microsomes (3 mg) with arachidonic acid in presence of NADPH and indomethacin. Separate and purified by reverse and normal phase liquid chromatography as described by Schwartzmann in 1988. For this rat aortic rings, male Sprague Dawley rats (300-350 g) were killed by cervical dislocation and thoracic aorta was carefully removed and placed into cold Kreb""s bicarbonate buffer freed of periadventitial fat and cut into 3-4 mm wide rings. To ensure the integrity of the vascular endothelium, care was taken during the dissection to avoid stetching or contact of instrument with the luminal surface of the ring. The aortic rings were mounted in 5 ml of water jacketed organ bath maintained at 37xc2x00 C. and equilibrated for 1.5-2 hrs. in Kreb""s bicarbonate buffer gassed with 95% O2 and 5% CO2. The composition of the Kreb""s bicarbonate buffer was (g/l) NaCl 6.95; KCl 0.354; CaCl2 0.280; KH2PO4O.162; MgSO4, 7 H2O 0.294, NaHCO, 2.1 and dextrose 2.0. A minimum of four rings was used simultaneously from each aorta. Basal tone was set at 2 g and adjusted accordingly over the equilibrium period. Tension was measured using glass model RPS 7C8xcex9. This procedure provided optimal conditions for reproducible isomatic force development.
There are always side products formed hence purification of the desired end product increases cost of the metabolite (Sigma price of 10 xcexcgs of 20 HETE is 78 $)
The main object of the present invention is to provide a novel process for preparation of a mixture of 19 hydroxyeicosatetraenoic acid and 20 hydroxyeicosatetraenoic acid (19 HETE and 20 HETE).
Another object of the invention is to provide one step transformation of arachidonic acid to a mixture of 19 HETE and 20 HETE as compared to the 10 step process of the prior art (Falck et al., 1988).
Still another object of the present invention is to provide a process for the hydroxylation which is stereo specific, in case of 19 HETE.
Yet another object of the present invention is to provide a process wherein the conversion efficiency of Arachidonic acid to 19 HETE and 20 HETE is 100 times higher than that of mammalian cells as there are only two metabolites formed and they are to metabolized further. In chemical as well as biological methods the yield is ver low and in picomoles whereas microbial transformation has shown in milligrams levels. Yet another object is to provide a process for preparation of mixture of 19 hydroxyeicosatetraenoic acid and 20 hydroxyeicosatetraenoic acid (19 HETE and 20 HETE) in which yeast cells require less restricted conditions and cheaper carbon sources such as molasses, cornsteep liquor etc., than mammalian cells or isolated P 450 from mammalian cells.
Yeast cells are grown in conventional medium, containing glucose as carbon source with moderate temperature at 30xc2x00 C. and other conditions, whereas in chemical methods for preparation of the same requires temperature ranging from xe2x88x9228 to 37xc2x0 C. In biological methods it requires absolutely restricted conditions as described in the prior art.
Accordingly, the present invention provides a process for the preparation of mixture of 19 hydroxyeicosatetraenoic acid and 20 hydroyeicosatetraenoic acid (19 HETE and 20 HETE) which comprises the steps of growing yeast species such as Canadian apicola (ATCC 96134, ATCC 24616) (Isolated from intestine of bee Ref: Antoinie Van Leeuwenhoek, 24 18, 1958) or Candida bombicola (ATCC 22214) (formerly known as Torulopsis bombicola isolated from Bumblebee honey. Ref: Agr. Biol. Chem, 44.221-2223, 1980 and Biotch.Lett 6,225-230, 1984) (i.e. yeast species) in a conventional growth medium consisting of carbon, nitrogen sources and other micro-ingredients supplemented with arachidonic acid or Poly Unsaturated Fatty Acids (PUFA) for a period of 12 to 96 hrs., in a known manner, separating the biomass from the medium (broth), by conventional methods, extracting the separated broth with an organic solvent, drying by conventional method, hydrolyzing by conventional method or by enzymatic hydrolysis to obtain 19 HETE and 20 HETE. These microorganisms used in the invention i.e. Candida apicola (ATCC 96134, ATCC 24616) or Candida bombicola (ATCC 22214) are publicly available without any difficulty and there is no difficulty in securing samples of these microorganisms.
In one of the embodiments of the present invention, arachidonic acid or an oil containing polyunsaturated fatty acids is added to the medium at the time of the inoculation of the yeast sp or added after a period of growth.
In another embodiment the Poly Unsaturated Faty Acids (PUFA) may be such as linoleic acid, alpha and gamma linolenic acid, dibromo gamma linolanic acid eicosapentaenoic, docosa pentaenoic acid (20:5, n-6 and 20:5, n-3) and docadthexacnoic acid.
In still another embodiment the concentration of the arachidonic acid or PUFA may be ranging between 10 mg to 5000 mg/lit of the medium either by single addition or by programmed additional (dose dependent).
In another embodiment the solvent used for supplementing of arachionic acid or PUFA may be alkanols preferably ethanol or by direct addition of arachidonic acid or by addition of methyl or ethyl esters of arachidonic acid.
In another embodiment the solvent used for extraction of the mixture of products may be polar solvents exemplified by but not limited to ethyl acetate, chloroform, n-hexane preferably ethyl acetate.
In a feature of the present invention the mixture of the 19 hydroxyelcosatetraenoic acid and 20 hydroxyeicosatetraenoic acid and 20 hydroxyeicosatetraenoic acids may be separated to free hydroxy fatty acids by acid hydrolysis effected by using mineral acid such as molar (dilute) HCL, H2 SO4 and nitric acid.
In a feature of the present invention, the mixture of the conjugated 19 hydroxyeicosatetraenoic acid and 20 hydroxyeicosatetraenoic acids may be separated to free fatty acids by enzymatic hydrolysis and effected by using xcex2-glucosidase or by similar enzymes.
In yet another feature of the present invention, the conventional media composition of the fermentor medium may be as given in Tables 1 and 2.
In yet another feature of the present invention the yeast used may be optionally immobilized by conventional methods like using calcium alginate. The use of immobilized yeast enables continuous production.
The process of the present invention is described hereinbelow with reference to examples, which are illustrative in nature and should not be construed to limit the scope of the present invention in any manner.